western blot mystery

Siddhartha S Mitra ssmitra at buffalo.edu
Wed Dec 8 11:49:04 EST 2004


criss.hartzell at emory.edu wrote:
> Sorry- my email address was not shown on the previous post.
> 
> I would appreciate suggestions on how to interpret the following result.
> I have SDS extracts from untransfected HEK293 cells and HEK293 cells
> transfected with cDNA for a protein called mB2. On westerns with mB2
> antibody, there is a nice band at the predicted 57kD in transfected cells
> and no band in untransfected cells. So far, it looks clean. However, if I
> add the SDS extracts from the untransfected cells to the extract from
> transfected cells, the intensity of the mB2 band decreases with increasing
> amounts of untransfected extract added (the gel is loaded with the same
> amount of transfected protein in each lane - the total amount of protein per
> lane is increased by addition of untransfected extract). At a ratio of 1:1,
> the mB2 band completely disappears. It is not due to proteolysis because the
> extracts have been heated to 100C for 10 min prior to mixing. The mB2 band
> is not migrating at a different place in the gel, because no signal is found
> either in the stacking gel or elsewhere. Adding BSA or serum to the
> transfected cell extract does not have the same effect except at very high
> concentrations. Has anyone seen anything like this before?
> 
> Criss Hartzell
> 
> 
> 
Is there any changes if you simply commassie or silver stain your gel????



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